PROJECT SUMMARY Human norovirus (HuNoV) gastroenteritis is a significant public health burden worldwide. The lack of a robust and reproducible HuNoV cell culture system still limits our ability to study fundamental aspects of HuNoV infection. While several recent breakthroughs increased our ability to propagate HuNoVs in vitro (e.g. B cell and human enteroid cultures), these systems are not robust enough, can be time consuming and expensive or hard to replicate. The surrogate models available for HuNoV research do not necessarily reflect essential biological features of HuNoVs and their natural host. In this proposal we will use our novel rhesus enteric caliciviruses (ReCV) model that reflects the biological features and diversity of HuNoVs to identify host determinants of infection. Zoonotic/interspecies transmission of ReCVs and HuNoVs between human and non- human primate hosts suggests evolutionary conservation of shared factors of host susceptibility between the two genera. Here we seek to identify host determinants of enteric calicivirus infection using CRISPR-Cas9 genome wide screening. We recently identified a functional ReCV entry receptor that is necessary for ReCV permissiveness in cell culture. We hypothesize that the ReCV entry receptor is also involved in HuNoV infections. This hypothesis will be tested in the following specific aims. Aim 1. Characterize receptor mediated ReCV infection. Aim 2: Comparative characterization of HuNoV and ReCV infections in enteroid and B cell cultures. In the first aim we will dissect the role of HBGAs in ReCV receptor mediated entry by using ReCV isolates with disctinct HBGA binding, cell lines expressing different HBGAs and/or the receptor, Enterobacter SENG-6 EPS and different synthetic HBGAs. We will also evaluate the role of the different transmembrane isoforms of the receptor in infection and map the ReCV interaction site and importance of glycosylation. In the second aim, we will evaluate the role of ReCV entry receptor in HuNoV infections in enteroid and B cell cultures and identify other cell surface components playing a role in HuNoV infections. We will also evaluate the mechanism of bile or bile salts in promoting HuNoV infections and the role of M cells in infections of polarized cells. Our long-term goal is to identify and characterize viral and host determinants of ReCV and HuNoV infections and to develop novel intervention/prevention strategies. Our proposal uses a novel enteric calicivirus model to understand viral entry and the role of bile and HBGAs in enteric viral infections. Our findings with ReCVs may be directly transferable to HuNoV infection. The major significance of this project is the identification of determinants of susceptibility to enteric calicivirus infection that can lead to improved HuNoV cell culture systems and new intervention/prevention strategies.